1. Field of the Invention
This invention relates to methods and apparatus for selectively targeting specific cells within a population of living cells. In particular, this invention relates to high-speed methods and apparatus for selectively identifying, and individually targeting with an energy beam, specific cells within a cell population to induce a response in the targeted cells.
2. Description of the Related Art
The use of cellular therapies is growing rapidly, and is therefore becoming an important therapeutic modality in the practice of medicine. Unlike other therapies, cellular therapies achieve a long-lasting, and often permanent benefit through the use of living cells. Hematopoietic stem cell (HSC) (e.g., bone marrow or mobilized peripheral blood) transplantation is one example of a practiced, insurance-reimbursed cellular therapy. Many other cellular therapies are being developed, including immunotherapy for cancer and infectious diseases, chondrocyte therapy for cartilage defects, neuronal cell therapy for neurodegenerative diseases, and stem cell therapy for numerous indications. Many of these therapies require the removal of unwanted, detrimental cells for full efficacy to be realized.
Gene therapy is another active area of developing medicine that can influence the success of cellular therapy. Given the rapid advances in the understanding of the human genome, it is likely that many genes will be available for insertion into cells prior to transplantation into patients. However, obtaining efficient targeted delivery of genes into specific cells of interest has remained a difficult obstacle in the development of these therapies.
In the treatment of cancer, it has been found that high-dose chemotherapy and/or radiation therapy can be used to selectively kill rapidly dividing cancer cells in the body. Unfortunately, several other cell types in the body are also rapidly dividing, and in fact, the dose-limiting toxicity for most anti-cancer therapies is the killing of HSCs and progenitor cells in the bone marrow. HSC transplantation was developed as a therapy to rescue the hematopoietic system following anti-cancer treatments. Upon infusion, the HSCs and progenitor cells within the transplant selectively home to the bone marrow and engraft. This process is monitored clinically through daily blood cell counts. Once blood counts return to acceptable levels, usually within 20 to 30 days, the patient is considered engrafted and is released from the hospital.
HSC transplants have been traditionally performed with bone marrow, but mobilized peripheral blood (obtained via leukapheresis after growth factor or low-dose chemotherapy administration) has recently become the preferred source because it eliminates the need to harvest approximately one liter of bone marrow from the patient. In addition, HSCs from mobilized peripheral blood result in more rapid engraftment (8 to 15 days), leading to less critical patient care and earlier discharge from the hospital. HSC transplantation has become an established therapy for treating many diseases, such that over 45,000 procedures were performed worldwide in 1997.
HSC transplantation may be performed using either donor cells (allogeneic), or patient cells that have been harvested and cryopreserved prior to administration of high-dose anti-cancer therapy (autologous). Autologous transplants are widely used for treating a variety of diseases including breast cancer, Hodgkin""s and non-Hodgkin""s lymphomas, neuroblastoma, and multiple myeloma. The number of autologous transplants currently outnumbers allogeneic transplants by approximately a 2:1 ratio. This ratio is increasing further, mainly due to graft-versus-host disease (GVHD) complications associated with allogeneic transplants. One of the most significant problems with autologous transplants is the reintroduction of tumor cells to the patient along with the HSCs, because these tumor cells contribute to relapse of the original disease.
As a tumor grows, tumor cells eventually leave the original tumor site and migrate through the bloodstream to other locations in the body. This process, called tumor metastasis, results in the formation and growth of satellite tumors that greatly increase the severity of the disease. The presence of these metastatic tumor cells in the blood and other tissues, often including bone marrow, can create a significant problem for autologous transplantation. In fact, there is a very high probability that metastatic tumor cells will contaminate the harvested HSCs that are to be returned to the patient following anti-cancer therapy.
The presence of contaminating tumor cells in autologous bone marrow and mobilized peripheral blood harvests has been confirmed in numerous scientific studies. Recent landmark studies have unambiguously shown that reinfused tumor cells do indeed contribute to disease relapse in humans (Rill et al. 1994). This was proven by genetically marking the harvested cells prior to transplant, and then showing that the marker was detected in resurgent tumor cells in those patients who relapsed with disease. These data have been confirmed by other investigators (Deisseroth et al. 1994), indicating that contaminating tumor cells in HSC transplants represent a real threat to patients undergoing autologous transplantation.
Subsequent detailed studies have now shown that the actual number of tumor cells reinfused in the transplant was correlated with the risk of relapse for acute lymphoblastic leukemia (Vervoordeldonk et al. 1997), non-Hodgkin""s lymphoma (Sharp et al. 1992; Sharp et al. 1996), mantle cell lymphoma (Andersen et al. 1997), and breast cancer (Brockstein et al. 1996; Fields et al. 1996; Schulze et al. 1997; Vannucchi et al. 1998; Vredenburgh et al. 1997). One of these studies went even further, showing that the number of tumor cells infused was inversely correlated with the elapsed time to relapse (Vredenburgh et al. 1997). These data suggest that reducing the number of tumor cells in the transplant will lead to better outcomes for the patient.
Due to the known risk of tumor cell contamination in autologous transplantation, a number of methods have been proposed for removing contaminating tumor cells from harvested HSC populations. The basic principle underlying all purging methods is to remove or kill tumor cells while preserving the HSCs that are needed for hematopoietic reconstitution in the patient.
One such method utilized fluorescence-activated cell sorting (FACS) to sort HSCs from tumor cells (Tricot et al. 1995). As is known, flow cytometry sorts cells one at a time and physically separates one population of cells from a mixture of cells based upon cell surface markers and physical characteristics. However, it has been shown that using FACS to separate large cell populations for clinical applications is not advantageous because the process is slow, the cell yields can be very low, and purity greater than xcx9c98% is rarely achieved.
Another method utilizing a flow cytometer is described in U.S. Pat. No. 4,395,397 to Shapiro. In the Shapiro method, labeled cells are placed in a flow cytometer, and a downstream laser beam is used to kill the labeled cells in the flowing stream after they pass by the detector and are recognized as being labeled by the electronic system. This method suffers from a number of disadvantages. Firstly, once an unwanted cell has passed through the detector/laser region there is no way to check that destruction has been completed successfully. If a tumor cell evades destruction it will inevitably be reintroduced into the patient. Secondly, the focal spot diameter of the laser beam is of necessity greater than the liquid stream cross section. Accordingly, many of the HSCs in the region of an unwanted cell will also be destroyed by the laser beam. Also, as described above, the purity obtained by flow cytometric techniques is not very good due to the random and dynamic nature of a heterogeneous cell mixture that is flowing in a fast-moving (1-20  m/sec) stream of liquid.
Another method that utilizes laser technology is described in U.S. Pat. No. 4,629,687 to Schindler, et al. In this method, anchorage-dependent cells are grown on a movable surface, and then a small laser beam spot is scanned across the moving surface to illuminate cells one at a time and the information is recorded. The same laser is then switched to a higher lethal power level, and the beam is swept over the surface in all areas except where a cell of interest was recorded during the illumination step. Unfortunately, this method is slow and only will work on cells that can adhere to a surface.
A still further method that utilizes laser technology is described in U.S. Pat. No. 5,035,693 to Kratzer. In this method, cells are placed on a moving belt and a small laser beam spot is scanned across the surface. When a particular cell radiates in response to the illuminating laser spot, the same laser is quickly switched to high power in order to kill the cell in a near simultaneous manner before the scanner has moved appreciably away from that cell. However, this system has many of the same disadvantages as the Shapiro method. For example, because the scanner is continuously moving during the imaging and killing of cells, the system is highly-dynamic, and therefore less stable and less accurate than a static system. Also, because the cells are moving on a belt past the detector in one direction, the method is not reversible. Thus, if a single tumor cell escapes detection, it will be reintroduced into the patient.
Others have used a small laser beam spot to dynamically scan over a surface to illuminate cells. For example, U.S. Pat. No. 4,284,897 to Sawamura et al. describes the use of galvanometric mirrors to scan a small laser beam spot in a standard microscope to illuminate fluorescent cells. U.S. Pat. No. 5,381,224 to Dixon et al. describes imaging of macroscopic specimens through the use of a laser beam spot that is raster-scanned with galvanometric mirrors through an F-theta scanning lens. In U.S. Pat. Nos. 5,646,411, 5,672,880, and 5,719,391 to Kain, scanning of a small laser spot with galvanometers through an F-theta lens is described. All of these imaging methods dynamically illuminate a small point that is moved over the surface to be imaged. In some cases, the surface being scanned is also moving during imaging.
Similar methods of scanning a small laser spot have been described for purposes other than imaging of cells. For example, U.S. Pat. No. 4,532,402 to Overbeck describes the use of galvanometers to move a small laser beam spot over a semiconductor surface for repair of an integrated circuit. Similarly, U.S. Pat. No. 5,690,846 to Okada et al. describes laser processing by moving a small laser spot with mirrors through an F-theta scanning lens. U.S. Pat. No. 5,296,963 to Murakami et al. describes the use of galvanometric mirrors to scan a small laser beam spot in a standard inverted microscope to puncture cells for insertion of genetic matter.
Yet another method of scanning a biological specimen is described in U.S. Pat. No. 5,932,872 to Price. This method uses a plurality of detectors to simultaneously capture images at a plurality of focus planes from a constantly moving surface. The resultant images can be used to choose the best-focus image in real-time, and can be used to generate a three-dimensional volumetric image of a specimen.
Most of the methods described above are based on administering a tumor cell-removal or tumor cell-killing strategy to the entire harvested cell population as a whole. In flow cytometry, cells are sorted on a single cell basis to physically separate the unwanted tumor cells from HSCs. While each of these methods has been shown to reduce tumor cell numbers in HSC transplants, none has demonstrated the ability to remove or kill all detectable tumor cells. In fact, the majority of patient transplants still contain detectable tumor cells after these purging techniques are used. Approximately 30 to 30,000 tumor cells per transplant still remain, even after multiple-step purging procedures (Gazitt et al. 1995; Gribben et al. 1991; Mapara et al. 1997; Paulus et al. 1997). Further, all of these methods result in some degree of HSC loss or damage, which can significantly impact the success of the HSC transplant by delaying patient engraftment. In summary, existing purging technologies are inadequate, and there exists a great unmet clinical need for novel approaches that can effectively purge all detectable tumor cells from an HSC transplant. The method and apparatus described herein fulfills this need.
High throughput screening for the action of candidate drug compounds on biological specimens is another area of great importance. Typically, a large number of candidate compounds is applied in parallel to small cell samples placed in wells of a multi-well plate, and each well is examined for some change in a biological indicator. Due to the large number of compounds, speed of screening is an important factor. Such studies are currently limited by capturing a signal from the cell population as a whole, or by laborious manual viewing of individual cells with microscopes. The former precludes the possibility of observing an effect on a cell subpopulation or of observing an effect within only a portion of the cell, whereas the latter approach is too slow to apply to numerous candidate compounds. An apparatus and method that could rapidly measure the effect of candidate compounds on individual cells is in great need.
This invention provides a high-speed method and apparatus for selectively identifying, and individually targeting with an energy beam, specific cells within a cell population for the purpose of inducing a response in the targeted cells. Using the apparatus of the present invention, every detectable target cell in a cell population can be specifically identified and targeted, without substantially affecting cells that are not being targeted. The cells can be a mixed population or of a homogenous origin.
Specific cells are identified with the disclosed invention using several approaches. One embodiment includes a non-destructive labeling method so that all of the cells of a first population are substantially distinguishable from the remaining cells of the cell mixture, the remaining cells comprising the second population. In this embodiment, a labeled antibody can be used to specifically mark each cell of the first population, yet not mark cells of the second population. The labeled cells are then identified within the cell mixture. A narrow energy beam is thereafter focused on the first of the targeted cells to achieve a desired response. The next of the targeted cells is then irradiated, and so on until every targeted cell has been irradiated.
In another embodiment, an antibody that selectively binds to cells of the second population, but not cells of the first population, is used to identify cells of the first population. Cells of the first population are identified by the absence of the label, and are thereafter individually targeted with the energy beam.
The nature of the response that is induced by the energy beam is dependent upon the nature of the energy beam. The response can be lethal or non-lethal. Thus, examples of responses that can be induced with an energy beam include necrosis, apoptosis, optoporation (to allow entry of a substance that is present in the surrounding medium, including genetic material), cell lysis, cell motion (laser tweezers), cutting of cell components (laser scissors), activation of a photosensitive substance, excitation of a fluorescent reagent, photobleaching, and molecular uncaging.
Another aspect of the invention is directed to an apparatus for determining a morphological or physiological characteristic of individual cells in a biological specimen. Thus, the apparatus would directly or indirectly induce a non-lethal response in a population of cells, and measure the response. Such non-lethal responses include optoporation, cell motion (laser tweezers), cutting of cell components (laser scissors), activation of a photosensitive substance and excitation of a fluorescent reagent. Such non-lethal responses may also include the responses of cells targeted by the techniques of photobleaching, such as photobleaching recovery, and molecular uncaging, both internal and external with respect to the cell. An example of such apparatus would comprise:
An illumination source for illuminating a frame of cells in said biological specimen;
An image capture system that captures an image of said frame of cells;
First commands for determining the location of a first individual cell in said biological specimen by reference to said image;
An energy source that emits an energy beam sufficient to induce a response in at least one or more individual cells;
Second commands for intersecting said first individual cell with an energy beam sufficient to interrogate said first individual cell for the presence of a morphological or physiological characteristic; and
A detector for measuring the response of said first individual cell to said interrogation.
Alternatively, such an apparatus would comprise:
An illumination source for illuminating a frame of cells in said biological specimen;
An image capture system that captures an image of said frame of cells;
First commands for determining the location of a first targeted cell in said biological specimen by reference to said image;
Second commands for intersecting said first targeted cell with an energy beam sufficient to cause a change of a morphological or physiological characteristic in said first targeted cell; and
A detector for measuring the response for the change in said morphologic or physiological characteristic of said first targeted cell.
Another aspect of the invention is an apparatus an illumination source for illuminating a frame of cells in said biological specimen comprising, an image capture system that captures an image of said frame of cells, first commands for determining the location of an individual cell in said biological specimen by reference to said image, an energy source that emits an energy beam sufficient to induce a response in at least one of the individual cells, second commands for intersecting said first individual cell with an energy beam sufficient to cause a change of a morphological or physiological characteristic in said first individual cell, and a detector for measuring a morphological or physiological characteristic of said first individual cell.
Another aspect of the invention is an apparatus for determining a morphological or physiological characteristic of individual cells in a biological specimen comprising:
an illumination source for illuminating a frame of cells in said biological specimen, wherein said specimen contains a quantity of caged compounds;
an image capture system that captures an image of said frame of cells;
first commands for intersecting one or more of said quantity of caged compounds with an energy beam sufficient to uncage said one or more of said quantity of caged compounds; and
an energy source that emits an energy beam sufficient to uncage one or more of said quantities of caged compounds;
second commands to monitor the progress of the change in said morphological or physiological characteristic in response to said uncaged compounds of said one or more of said quantity of caged compounds.
Another such apparatus for determining a morphological or physiological characteristic of individual cells in a biological speciment comprises:
an illumination source for illuminating a frame of cells in said biological specimen;
an image capture system that captures an image of said frame of cells; and
first comands for determining the morphological or physiological characteristic of said individual cell in said frame of cells.
The above apparatus can be used for high throughput screening of responses of cells to outside stimuli, in that it provides rapid location and measurement of responses of individual cells to such stimuli. Thus, the illumination source and/or the energy beam could target molecules in the vicinity of the cells, whether or not the molecules are in or on the cells, the energy beam could interact with and activate the molecules and the illumination source could study the response of the cells to the molecules so activated.
A large number of commercially important research and clinical applications can be envisioned for such an apparatus, examples of which are presented below.